Insect ecdysis sequences represent a simple, robust, and tractable model for studying the neuromodulatory mechanisms that govern behavior. Because initiation of an ecdysis sequence involves a profound shift in behavioral priorities, study of these sequences offers the opportunity to understand the neuromodulatory mechanisms that govern changes in behavioral state. In addition, because ecdysis behaviors are inherently sequential, they permit the systematic investigation of how motor programs are assembled and serially executed by the nervous system. Finally, the study of ecdysis sequences promises insight into how conserved circuits can be variably configured to generate immensely different behaviors. In Drosophila, for example, the motor sequences performed at pupal and adult ecdysis before and after metamorphosis, respectively are scarcely similar though they are governed by a common set of neuromodulatory/hormonal inputs. By analogy to computing, these inputs can be regarded as instructions written in a higher programming language that are then compiled into different motor output patterns. Exposing the mechanisms of neural compilation in ecdysis is likely to deeply inform our understanding of how neuromodulators contribute to neurocomputation by reconfiguring the activity of neural networks. To investigate these questions, our laboratory seeks to elucidate the circuitry that governs both the pupal and adult ecdysis sequences in Drosophila, though during the past year, we have focused primarily on the neuronal determinants of pupal ecdysis. As noted above, pupal ecdysis is strictly dependent on intrinsic factors, a fact most evident from the observation that exogenous application of the peripherally released hormone, Ecdysis Triggering Hormone (ETH) to an isolated pupal nervous system is sufficient to trigger sequential activation of neurons that express other key neuromodulators governing ecdysis. Demonstrating that this sequential activation correlates with the generation of a generalized fictive ecdysis sequence is a goal of our current work, and our efforts over the last year have focused on identifying and characterizing the direct neuronal targets of ETH. To identify these targets, we have used the Trojan exon technique introduced by our laboratory in a major paper from the previous year (Diao et al., 2015, Cell Reports 10, 14101421). Trojan exons enable the identification and genetic manipulation of neurons that express a specific gene of interest and we have used this tool to selectively target neurons that express each of the two isoforms of the ETH receptor/ gene, ETHRA and ETHRB. As described in a paper published this year (Diao et al., 2016, Genetics 202:175-189), we have shown that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. Our results from both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval ecdysis, and they further confirm the requirement at pupal ecdysis for ETHRA-expressing neurons that co-express the hormone Crustacean Cardioactive Peptide (CCAP). In this work, we also identified other functionally important subsets of ETHRA-expressing neurons including one that co-expresses the peptide Leucokinin. We demonstrated that Leucokinin-expressing neurons regulate fluid balance to facilitate ecdysis at the pupal stage. Our recent work thus augments our understanding of neurons that act downstream of ETH to mediate the pupal ecdysis sequence. As noted, these neurons include cells that express other hormones critical for generating the pupal ecdysis sequence, such as CCAP. Identification and functional characterization of the neuronal targets of CCAP and other hormones is the goal of current work in the laboratory. This work also relies on the Trojan exon method and our success in applying this technique to elucidating the pupal ecdysis circuit illustrates a general strategy for mapping hormonally regulated networks. Using a receptor gene as an entry point for genetic and neuronal manipulations, one can establish patterns of functional connectivity between neurons at different levels in the network. In summary, we have made clear progress during the last year in elucidating the developmentally dynamic circuit in the Drosophila nervous system that supports ecdysis. Our work continues to uncover neuronal substrates of ecdysis behavior and complements other research on the functional architecture of the Drosophila nervous system. As argued in an invited perspective published during the last year (White, B.H., 2016, J Neurogenet: 30:54-61), intensive investigation of nervous system organization in Drosophila and other genetic model animals promises a comprehensive and hitherto unprecedented understanding of how nervous systems function to arbitrate an animals needs and desires in the course of a lifetime and should deliver indispensable insights into the general operation of all nervous systems. These insights should thus shed light on the deficits in behavioral organization that lie at the root of many mental disorders.